Abstract Purpose Previous research has demonstrated increased stiffness in the multifidus muscle compared to other paraspinal muscles at the fiber bundle level. We aimed to compare single fiber and fiber bundle passive mechanical properties of multifidus muscle: (1) in 40 patients undergoing primary versus revision surgery and (2) in muscle with mild versus severe fatty infiltration. Methods The degree of muscle fatty infiltration was graded using the patients' spine magnetic resonance images. Average single fiber and fiber bundle passive mechanical properties across three tests were compared between primary (N = 30) and revision (N = 10) surgery status, between mild and severe fatty infiltration levels, between sexes, and with age from passive stress–strain tests of excised multifidus muscle intraoperative biopsies. Results At the single fiber level, elastic modulus was unaffected by degree of fatty infiltration or surgery status. Female sex (p = 0.001) and younger age (p = 0.04) were associated with lower multifidus fiber elastic modulus. At the fiber bundle level, which includes connective tissue around fibers, severe fatty infiltration (p = 0.01) and younger age (p = 0.06) were associated with lower elastic modulus. Primary surgery also demonstrated a moderate, but non‐significant effect for lower elastic modulus (p = 0.10). Conclusions Our results demonstrate that female sex is the primary driver for reduced single fiber elastic modulus of the multifidus, while severity of fatty infiltration is the primary driver for reduced elastic modulus at the level of the fiber bundle in individuals with lumbar spine pathology.